Pancreatic cancer (PC) has the highest mortality of any cancer, with the 5-year survival rate less than 6%. Although there has been some progress in the use of improved diagnostic methods and development of targeted therapies, the overall survival rate has not improved over the last decade. It is therefore important to identify novel molecular markers and therapeutic targets in PC that could lead to more effective treatment for this malignant disease. The novel concept in this proposal is that a zinc transporter, ZIP4, regulates PC growth and survival, which assigns a new and critical role for ZIP4. We have shown that ZIP4 is overexpressed in a majority of PC patients (>80%) and contributes to PC pathogenesis, indicating that ZIP4 is a promising prognostic marker and therapeutic target. However, how does ZIP4 regulate PC is not clear. In order to further understand the molecular mechanisms of ZIP4-mediated PC growth, we investigated whether certain microRNAs (miRNAs) as well as their target genes are regulated by ZIP4 in PC. We examined the expression of 95 miRNAs in ZIP4-overexpressed or silenced cells and xenografts. We found that the expression of several miRNAs such as miR-373 positively correlates with the ZIP4 level, while other miRNAs such as miR-122a inversely correlates with ZIP4. We are particularly interested in miR- 373 for the following reasons: 1) miR-373 is the most upregulated miRNA upon ZIP4 overexpression with the highest fold change compared with other miRNAs, and 2) there is a strong correlation between miR-373 and ZIP4 level in every pairwise comparison in both ZIP4-overexpressed or silenced cells and xenografts. Moreover, miR-373 is an oncogenic miRNA in breast cancer and testicular germ-cell tumors. Therefore, we chose miR-373 for further analysis to expand our understanding of the mechanism of ZIP4-mediated PC growth. Our preliminary data demonstrate that miR-373 is upregulated by ZIP4 through cAMP response element-binding protein (CREB)-mediated transcriptional activation. Potential target genes of miR-373, TP53-inducible nuclear protein 1 (TP53INP1) and large tumor suppressor homolog 2 (LATS2), which are predicted by three different miRNA target prediction programs, are found to be repressed by ZIP4 and miR- 373 in PC. Importantly, the two target genes were also shown as tumor suppressors in PC. In this proposal we will use these findings as a foundation to further our understanding of the mechanism underlying ZIP4 oncogenic function in PC. We hypothesize that ZIP4 promotes PC growth through a distinct signaling pathway leading to the activation of a CREB-miR-373 axis and the expression of this ZIP4-miR-373 pathway could serve as a prognostic marker for this dismal disease. Three independent but interrelated specific aims are proposed to address this hypothesis. We will determine the prognostic/predictive values of ZIP4 and miR-373 expression in human PC. We will study the signaling pathway downstream of ZIP4 activating CREB-dependent transcription of miR-373 in PC. Finally, we will investigate the epigenetic mechanism by which CREB controls miR-373 expression downstream of ZIP4 in PC. ZIP4-induced CREB- miR-373 upregulation represents a novel signaling pathway modulating the growth of PC tumors. The proposed studies will help define the mechanism regulating this oncogenic axis and determine the prognostic/predictive value of the ZIP4-miR-373 axis in PC growth. The results obtained from this study will be of high impact since it will shed light on future development of diagnostic and therapeutic strategies in PC. Thus, this proposal is mechanistic and translational in nature.